七氟烷致神经元损伤的机制:Tau蛋白磷酸化与含18 kDa片段的载脂蛋白E的关系

Mechanism of neuronal damage induced by sevoflurane: the relationship between phosphorylation of Tau protein and apolipoprotein E containing 18 kDa fragments

目的评价Tau蛋白磷酸化与含18 kDa片段的载脂蛋白E(ApoE)的关系,探讨七氟烷致神经元损伤的机制。方法将培养至第5天的ApoE3型和ApoE2型人源化胎鼠原代神经元,各24皿,分别采用随机数字表法分为4组(n=12):ApoE3对照组(A3C组)、ApoE3七氟烷组(A3S组)、ApoE2对照组(A2C组)和ApoE2七氟烷组(A2S组)。A3S组和A2S组给予21%氧气+5%二氧化碳+4.1%七氟烷连续4 h处理,A3C组和A2C组只给予21%氧气+5%二氧化碳处理。随后提取细胞蛋白采用Western blot法检测全长ApoE、含18 kDa片段的ApoE、AT8和PHF1表达,RT-PCR法检测ApoE mRNA表达,ELISA法检测上清液TNF-α及IL-6浓度。结果与A2C组比较,A2S组神经元ApoE mRNA和全长ApoE表达上调(P<0.05),AT8和PHF1表达、上清液TNF-α和IL-6浓度差异无统计学意义(P>0.05);与A3C组比较,A3S组神经元ApoE mRNA、全长ApoE、含18 kDa片段的ApoE、AT8和PHF1表达上调,上清液TNF-α和IL-6浓度升高(P<0.05)。结论七氟烷可能通过上调含18 kDa片段的ApoE表达,促进Tau蛋白磷酸化,增加炎症反应,导致神经元损伤。

Objective To evaluate the relationship between phosphorylation of Tau protein and apolipoprotein E(ApoE)containing 18 kDa fragments and investigate the mechanism of neuronal damage induced by sevoflurane.Methods Primary neurons(ApoE3 and ApoE2 genotypes,24 dishes for each genotype)of fetal mice cultured until the 5th day were divided into 4 groups(n=12 each)using a random number table method:ApoE3 control group(A3C group),ApoE3 sevoflurane group(A3S group),ApoE2 control group(A2C group)and ApoE2 sevoflurane group(A2S group).Neurons were treated with 21%oxygen+5%carbon dioxide+4.1%sevoflurane for 4 h in A3S and A2S groups,while the neurons were only treated with 21%oxygen+5%carbon dioxide in A3C and A2C groups.The cell proteins were then extracted to detect the expression of full-length ApoE and ApoE,AT8 and PHF1 containing 18 kDa fragments(by Western blot),expression of ApoE mRNA(by real-time polymerase chain reaction),and concentrations of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)in the supernatant(by enzyme-linked immunosorbent assay).Results Compared with A2C group,the expression of ApoE mRNA and full-length ApoE in neurons was up-regulated(P<0.05),and no significant change was found in the expression of AT8 and PHF1 and concentrations of TNF-αand IL-6 in the supernatant in A2S group(P>0.05).Compared with A3C group,the expression of ApoE mRNA,full-length ApoE,and ApoE,AT8 and PHF1 containing 18 kDa fragments was up-regulated,and the concentrations of TNF-αand IL-6 in the supernatant were increased in A3S group(P<0.05).Conclusion Sevoflurane may promote phosphorylation of Tau proteins and increase inflammatory responses through up-regulating the expression of ApoE containing 18 kDa fragments,thus leading to neuronal damage.